The P-type (or E1E2) ATPases undergo an extraordinary series of conformational changes during the course of their highly efficient transport cycles. Detailed structural studies of the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) and the Na++K+-ATPase (Na/K pump) in conditions that put the proteins into different chemically-defined intermediate states of the reaction cycle revealed important changes in the tertiary structures of the proteins both in the three major cytoplasmic domains and the transmembrane segments that comprise the ion transport sites. In this proposal, we address the conformational dynamics of the SERCA and Na/K pumps as they transition between intermediate states throughout their transport cycle. We will carry out the following specific aims: (1) correlate the dynamics between conformational states in the Na/K pump produced by voltage using site-directed fluorimetry, including missense incorporation of fluorescent unnatural amino acids in oocyte expression; (2) carry out a series of double-mutant cycle analyses of SERCA based on the predictions of non-native contacts identified by the molecular dynamics string method simulations of the transitions between chemically defined states; and (3) use single particle cryo-EM to evaluate the conformational heterogeneity of both Na/K pump and SERCA in different conformational states that are in different chemically defined catalytic states or transitioning between states. To facilitate such structural studies, we will create conformational specific single antigen binders to help identify the different domains.